JP4990606B2 - Lactic acid bacteria-containing feed - Google Patents
Lactic acid bacteria-containing feed Download PDFInfo
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- JP4990606B2 JP4990606B2 JP2006340029A JP2006340029A JP4990606B2 JP 4990606 B2 JP4990606 B2 JP 4990606B2 JP 2006340029 A JP2006340029 A JP 2006340029A JP 2006340029 A JP2006340029 A JP 2006340029A JP 4990606 B2 JP4990606 B2 JP 4990606B2
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/87—Re-use of by-products of food processing for fodder production
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Description
本発明は、特定の乳酸菌を含有する飼料に関する。 The present invention relates to a feed containing a specific lactic acid bacterium.
現在の畜産業においては、経営の効率化のため、食肉の生産性向上が図られている。その一環として、多頭化飼育が試みられているが、飼育密度の増加に伴い、飼育環境が劣化するため、家畜の健康が害されることが多くなる。家畜の疾病予防のために、抗生物質等の各種の動物用医薬品を添加した飼料が用いられているが、このような動物用医薬品等の多用には、耐性菌の出現や、食肉への医薬品の残存等の問題がある。そこで、家畜等にもともと備わっている生理機能を活性化させることにより、薬剤等に頼らず、家畜等の健康を維持し、安全な食肉を提供することが強く望まれている。 In the current livestock industry, meat productivity is being improved in order to improve management efficiency. As part of this, multi-headed breeding has been tried, but as the breeding density increases, the breeding environment deteriorates, and the health of livestock is often impaired. For the prevention of livestock diseases, feed containing various veterinary drugs such as antibiotics is used. For such veterinary drugs, the emergence of resistant bacteria and the drugs for meat There is a problem such as remaining. Therefore, it is strongly desired to provide safe meat by activating physiological functions inherent in livestock and the like, maintaining the health of livestock and the like without depending on drugs and the like.
家畜等の生理機能を活性化させるものとして、例えば、（１）ラクトバチルス（Ｌａｃｔｏｂａｃｉｌｌｕｓ）属及びロイコノストック（Ｌｅｕｃｏｎｏｓｔｏｃ）属に属する少なくとも１種以上の乳酸菌を含んでなることを特徴とする飼料添加物が開示されている（例えば、特許文献１参照。）。該飼料添加物は、整腸作用を有するため、家畜等の健康状態が改善されることに加え、飼料効率が良いため、糞尿の臭気を少なくし、飼育環境を改善する効果も有するものである。 For example, (1) Lactobacillus (Lactobacillus) genus and Leuconostoc genus (Leuconostoc) genus Lactobacillus, The thing is disclosed (for example, refer patent document 1). Since the feed additive has an intestinal regulating action, in addition to improving the health condition of livestock and the like, the feed additive has an effect of reducing the odor of manure and improving the breeding environment because of the high feed efficiency. .
その他、例えば、エンテロコッカス・フェカリス（Ｅｎｔｅｒｏｃｏｃｃｕｓ・ｆａｅｃａｌｉｓ）菌ＥＣ−１２株（コンビ社）を主原料とした殺菌乳酸菌入り混合飼料「ラクセルフォース」（兼松株式会社製）が市販されている。該混合飼料は、主に離乳期子豚の餌に混合し給餌することにより、下痢便の改善、感染症予防による離乳期子豚の死亡頭数減少等の効果が期待されるものである。該混合飼料の効果は、主原料であるエンテロコッカス・フェカリスの、生体内でのインターフェロン（ＩＦＮ）−γ産生能増強作用によるものと推測される。 In addition, for example, a mixed feed “Luxell Force” (manufactured by Kanematsu Co., Ltd.) containing sterilized lactic acid bacteria using, as a main raw material, Enterococcus faecalis EC-12 strain (Combi). The mixed feed is expected to have effects such as improvement of diarrheal stool and reduction of the number of dead piglets in the weaning piglet by preventing infection by mainly mixing and feeding with the feed of the weaning piglet. The effect of the mixed feed is presumed to be due to the in vivo enhancement of interferon (IFN) -γ production ability of Enterococcus faecalis, which is the main raw material.
ＩＦＮ−γは、抗ウィルス作用、抗腫瘍作用、免疫応答調節作用等を有しており、特に、マクロファージ、ナチュラルキラー（ＮＫ）細胞、細胞障害性Ｔ細胞等の免疫担当細胞の分化誘導・活性化に重要な役割を果たすものである。ＩＦＮの直接投与は、様々な副作用を引き起こすおそれがあるため、生体にもともと備わっているＩＦＮ−γ産生能を増強させることが好ましい。
しかしながら、上記（１）の飼料添加物では、整腸作用等により、家畜の健康向上に役立つものの、感染症等に対し、有効な予防法とは言い難い。また、上記の市販混合飼料は、感染症予防効果が期待されるものの、未だ効果が不充分である。 However, although the feed additive of (1) is useful for improving the health of livestock due to the intestinal regulating action and the like, it is difficult to say that it is an effective preventive method against infectious diseases. Moreover, although the above-mentioned commercially available mixed feed is expected to have an infectious disease preventing effect, the effect is still insufficient.
本発明は、従来になく高効率かつ安全に、家畜の免疫機能を向上させ得る飼料を提供することを目的とする。 An object of the present invention is to provide a feed that can improve the immune function of livestock in a highly efficient and safe manner.
本発明者らは、上記課題を解決すべく鋭意研究した結果、ロイコノストック・メセントロイデス（Ｌｅｕｃｏｎｏｓｔｏｃ・ｍｅｓｅｎｔｅｒｏｉｄｅｓ）に分類される新規乳酸菌が、ＩＦＮ−γ産生能増強効果を有すること、及び、該乳酸菌を含有する飼料により、従来になく高効率かつ安全に、家畜の免疫機能を向上させ得ることを見出し、本発明を完成させた。 As a result of intensive studies to solve the above problems, the present inventors have found that a novel lactic acid bacterium classified as Leuconostoc mesenteroides has an effect of enhancing IFN-γ production ability, and The present invention was completed by finding that the feed containing the lactic acid bacteria can improve the immune function of livestock with high efficiency and safety compared to conventional methods.
すなわち、本発明は、ロイコノストック・メセントロイデスＡＢＲＤ ＬｕＭ−５（ＦＥＲＭ Ｐ−２０５０１）を含有することを特徴とする飼料を提供するものである。
また、本発明は、前記ＡＢＲＤ ＬｕＭ−５を用いて発酵させたことを特徴とする飼料を提供するものである。
また、本発明は、前記ＡＢＲＤ ＬｕＭ−５を用いて、ビール粕を発酵させたことを特徴とする飼料を提供するものである。
また、本発明は、前記ＡＢＲＤ ＬｕＭ−５を用いて発酵させた発酵物を含有することを特徴とする飼料を提供するものである。
また、本発明は、前記ＡＢＲＤ ＬｕＭ−５を用いて、ビール粕を発酵させた発酵物を含有することを特徴とする飼料を提供するものである。
That is, this invention provides the feed characterized by containing Leuconostoc mescentroides ABRD LuM-5 (FERM P-20501).
Further, the present invention is to provide a feed, characterized in that fermented using the ABRD LuM-5.
Further, the present invention uses the ABRD LuM-5, there is provided a feed, characterized in that fermented beer lees.
Further, the present invention is to provide a feed characterized by containing the ABRD LuM-5 fermentations fermented with.
Further, the present invention uses the ABRD LuM-5, there is provided a feed characterized by containing a fermented product fermented beer lees.
本発明の飼料により、家畜の免疫機能を向上させ得るため、薬剤等に頼らず、家畜等の健康を維持し、安全な食肉を提供することができる。免疫機能向上効果が大きいため、従来品よりも少量で効果が期待でき、経済的にも好ましい。また、使用される乳酸菌ＡＢＲＤ ＬｕＭ−５（ＦＥＲＭ Ｐ−２０５０１）及びＡＢＲＤ ＬｕＭ−１（ＦＥＲＭ Ｐ−２０５００）は、共に漬け物から単離された乳酸菌であるため、副作用なく安全に家畜の飼料として使用できる。 Since the livestock immune function can be improved by the feed of the present invention, the health of livestock and the like can be maintained and safe meat can be provided without relying on drugs or the like. Since the effect of improving the immune function is great, the effect can be expected in a smaller amount than conventional products, which is economically preferable. Moreover, since lactic acid bacteria ABRD LuM-5 (FERM P-20501) and ABRD LuM-1 (FERM P-20500) used are both lactic acid bacteria isolated from pickles, they can be safely used as livestock feed without side effects. it can.
まず、本発明のロイコノストック・メセントロイデスに分類される乳酸菌ＡＢＲＤ ＬｕＭ−５（ＦＥＲＭ Ｐ−２０５０１）（以下、ＦＥＲＭ Ｐ−２０５０１と略記する）及びＡＢＲＤ ＬｕＭ−１（ＦＥＲＭ Ｐ−２０５００）（以下、ＦＥＲＭ Ｐ−２０５００と略記する）の分離手段及び菌学的性質を示す。 First, lactic acid bacteria ABRD LuM-5 (FERM P-20501) (hereinafter abbreviated as FERM P-20501) and ABRD LuM-1 (FERM P-20500) classified as Leuconostoc mescentroides of the present invention ( Hereinafter, separation means and mycological properties of FERMP P-20500) will be shown.
＜ＦＥＲＭ Ｐ−２０５００及びＦＥＲＭ Ｐ−２０５０１の分離手段＞
漬け物（白菜キムチ）から分離した乳酸菌の中から、ＩＦＮ−γ産生能の高い乳酸菌を選択することにより、ＦＥＲＭ Ｐ−２０５００及びＦＥＲＭ Ｐ−２０５０１を分離・同定した。具体的には、以下の方法により、分離することができる。
<Separation means for FERM P-20500 and FERM P-20501>
FERM P-20500 and FERM P-20501 were separated and identified by selecting lactic acid bacteria having high IFN-γ production ability from lactic acid bacteria isolated from pickles (Chinese cabbage kimchi). Specifically, it can be separated by the following method.
漬け物１０ｇに生理食塩水９０ｍｌを加え混合し、更に生理食塩水で段階的に希釈して乳酸菌選択培地（５％馬脱繊維素血液添加ＢＬ寒天培地、１．５％寒天含有ＭＲＳ培地、ＰＥＳ培地、Ｍ−Ｅ培地、Ｍｍａ−ＬＢＳ培地）に塗末し培養した。培養条件は、５％馬脱繊維素血液添加ＢＬ寒天培地及び１．５％寒天含有ＭＲＳ培地の場合には３０℃にて２日間、ＰＥＳ培地の場合には２０℃にて３〜４日間、Ｍ−Ｅ培地の場合には３７℃にて１日間、Ｍｍａ−ＬＢＳ培地の場合には３５℃にて９〜１０日間、いずれも嫌気条件下とした。菌の集落が形成されたら、別の同種の平板培地に画線塗布し同様に培養した。更に同じ操作を繰り返し、単一集落を形成する乳酸菌を分離した。分離した乳酸菌をそれぞれＭＲＳ培地に接種し、３０℃で一晩培養した。該培養液を波長６６０ｎｍでの吸光度（ＯＤ６６０）が１．０となるように調製し、ＭＲＳ培地に１／１００（ｖ／ｖ）（菌数：１０６ｃｅｌｌｓ／ｍＬ）の割合で接種した。３０℃で２４時間培養（本培養）し、生菌数約１０７〜１０９ｃｅｌｌｓ／ｍＬの培養液を得た。得られた培養液を８，０００ｒｐｍ（約１０，０００×ｇ）で２０分間の遠心分離をして集菌し、これを蒸留水で２回洗浄して菌体を得た。この菌体を、用いた液体培地量の１／１００量の蒸留水で懸濁し、１０５℃で１分間加熱して死菌体懸濁液を得た。該死菌体懸濁液を、凍結乾燥により乾燥処理し、死菌体菌末を得た。 90 g of physiological saline was added to 10 g of pickled vegetables, mixed, and further diluted stepwise with physiological saline to select lactic acid bacteria selection medium (BL agar medium supplemented with 5% horse defibrinated blood, MRS medium containing 1.5% agar, PES medium) , M-E medium, Mma-LBS medium). The culture conditions were BL agar medium supplemented with 5% horse defibrinated blood and MRS medium containing 1.5% agar for 2 days at 30 ° C., and PES medium for 3 to 4 days at 20 ° C. In the case of the M-E medium, anaerobic conditions were set for 1 day at 37 ° C., and in the case of the Mma-LBS medium, the conditions were set at 35 ° C. for 9 to 10 days. When fungal colonies were formed, streaks were applied to another plate medium of the same kind and cultured in the same manner. Further, the same operation was repeated to isolate lactic acid bacteria forming a single settlement. The separated lactic acid bacteria were each inoculated into MRS medium and cultured overnight at 30 ° C. The culture solution was prepared so that the absorbance at a wavelength of 660 nm (OD660) was 1.0, and inoculated into MRS medium at a rate of 1/100 (v / v) (bacteria count: 10 6 cells / mL). Culturing was performed at 30 ° C. for 24 hours (main culture) to obtain a culture solution having about 10 7 to 10 9 cells / mL of viable bacteria. The obtained culture solution was centrifuged at 8,000 rpm (about 10,000 × g) for 20 minutes to collect cells, which were washed twice with distilled water to obtain bacterial cells. This microbial cell was suspended in 1/100 of distilled water in the amount of the liquid medium used, and heated at 105 ° C. for 1 minute to obtain a dead cell suspension. The dead cell suspension was dried by lyophilization to obtain a dead cell powder.
次に、前記死菌体菌末のＩＦＮ−γ産生能を測定した。
まず、マウスから摘出した脾臓を、細胞培養液（ＡＩＭ−Ｖ、ＧＩＢＣＯ社製）を用いて洗浄・濾過した後、５×１０６ｃｅｌｌｓ／ｍＬの脾臓細胞浮遊液を調製した。該細胞浮遊液を９６穴平底培養プレートに２００μＬ／ウェルとなるように播いた後、それぞれのウェルに、最終濃度１００μｇ／ｍＬとなるように前記死菌体菌末を添加した。該９６穴平底培養プレートを、３７℃、５％ＣＯ２条件下で２日間培養した後、培養上清中のＩＦＮ−γ濃度をＥＬＩＳＡ法により測定した。前記死菌体菌末を添加しないコントロールの培養上清中のＩＦＮ−γ濃度と比較して、有意にＩＦＮ−γ濃度が高かった乳酸菌を選択することにより、ＦＥＲＭ Ｐ−２０５００及びＦＥＲＭ Ｐ−２０５０１を得た。ＥＬＩＳＡ法は、予め９６穴平底プレートに結合させる抗体としてａｎｔｉ−ｍｏｕｓｅ ＩＦＮ−γ抗体(ＣＡＬＴＡＧ ｌａｂｏｒａｔｏｒｉｅｓ社製)を、捕捉したＩＦＮ−γを検出するためにＢｉｏｔｉｎ ｒａｔ ａｎｔｉ−ｍｏｕｓｅ ＩＦＮ−γ抗体(ＢＤ Ｐｈａｒｍｉｎｇｅｎ社製)とＨＲＰ標識Ａｖｉｄｉｎ試薬（ｈｏｒｓｅｒａｄｉｓｈ ｐｅｒｏｘｉｄａｓｅ Ａｖｉｄｉｎ Ｄ，Ｖｅｃｔｏｒ社製)を、それぞれ用いて、常法により行った。
Next, the IFN-γ production ability of the dead cell body powder was measured.
First, the spleen extracted from the mouse was washed and filtered using a cell culture solution (AIM-V, manufactured by GIBCO), and then a spleen cell suspension of 5 × 10 6 cells / mL was prepared. The cell suspension was seeded on a 96-well flat-bottom culture plate at 200 μL / well, and the dead cell powder was added to each well to a final concentration of 100 μg / mL. The 96-well flat-bottom culture plate was cultured at 37 ° C. under 5% CO 2 for 2 days, and then the IFN-γ concentration in the culture supernatant was measured by ELISA. By selecting lactic acid bacteria having a significantly higher IFN-γ concentration compared to the IFN-γ concentration in the control culture supernatant to which the dead bacterial cell powder was not added, FERM P-20500 and FERM P-20501 Got. In the ELISA method, an anti-mouse IFN-γ antibody (manufactured by CALTAG laboratories) is used as an antibody to be bound to a 96-well flat bottom plate in advance, and a Biotin rat anti-mouse IFN-γ antibody (BD) is used to detect the captured IFN-γ. Pharmingen) and HRP-labeled Avidin reagent (horseradish peroxide Avidin D, Vector) were used in a conventional manner.
＜菌学的性質及び生理学的性質＞
本発明の新規乳酸菌株ＦＥＲＭ Ｐ−２０５００の１６ＳリボソームＲＮＡ遺伝子の塩基配列を配列表の配列番号１に示す。同じく新規乳酸菌ＦＥＲＭ Ｐ−２０５０１の１６ＳリボソームＲＮＡ遺伝子の塩基配列を配列表の配列番号２に示す。既知微生物の塩基配列のデータベース上で、これらの１６ＳリボソームＲＮＡ遺伝子の塩基配列について相同性検索を行ったところ、ＦＥＲＭ Ｐ−２０５００及びＦＥＲＭ Ｐ−２０５０１のいずれも、既知のロイコノストック・メセントロイデスに属する微生物の塩基配列と９９％の相同性を有していた。従って、本発明の乳酸菌はいずれもロイコノストック・メセントロイデスに属する微生物と同定された。なお、ＦＥＲＭ Ｐ−２０５００等の１６ＳリボソームＲＮＡ遺伝子の塩基配列は、常法により同定した。
<Mycological and physiological properties>
The base sequence of the 16S ribosomal RNA gene of the novel lactic acid strain FERM P-20500 of the present invention is shown in SEQ ID NO: 1 in the sequence listing. Similarly, the base sequence of the 16S ribosomal RNA gene of the novel lactic acid bacterium FERM P-20501 is shown in SEQ ID NO: 2 in the sequence listing. When a homology search was performed on the base sequences of these 16S ribosomal RNA genes on a database of base sequences of known microorganisms, both FERM P-20500 and FERM P-20501 were both known Leuconostoc mescentroides. And 99% homology with the base sequence of microorganisms belonging to. Therefore, all the lactic acid bacteria of the present invention were identified as microorganisms belonging to Leuconostoc mescentroides. The base sequence of 16S ribosomal RNA gene such as FERM P-20500 was identified by a conventional method.
また、相同性が９９％であり、完全に一致している既知微生物が検索されなかったことから、新規な乳酸菌であることが確認された。 Moreover, since the homologue was 99% and a known microorganism that was completely matched was not searched for, it was confirmed to be a novel lactic acid bacterium.
さらに、ＦＥＲＭ Ｐ−２０５０１及びＦＥＲＭ Ｐ−２０５００の死菌体粉末は、公知のロイコノストック・メセントロイデスに属する乳酸菌の死菌体粉末よりも、マウスから摘出した脾臓細胞に対するＩＦＮ−γ産生能増強効果が明らかに強いことが判明した。 Furthermore, the dead cell powders of FERM P-20501 and FERM P-20500 are more capable of producing IFN-γ for spleen cells extracted from mice than the known dead cell powders of lactic acid bacteria belonging to known Leuconostoc mescentroides. It was found that the enhancement effect was clearly strong.
ＦＥＲＭ Ｐ−２０５０１等は、いずれも公知のロイコノストック属乳酸菌と同様に、公知の培地を用いて、常法により培養することができる。公知の培地として、例えば、ＭＲＳ培地等がある。培養温度は、２０〜３７℃が好ましい。 FERM P-20501 and the like can be cultured by a conventional method using a known medium in the same manner as known leuconostock lactic acid bacteria. Examples of known media include MRS media. The culture temperature is preferably 20 to 37 ° C.
本発明の飼料は、ロイコノストック・メセントロイデスに分類される乳酸菌ＦＥＲＭ Ｐ−２０５０１又はＦＥＲＭ Ｐ−２０５００を含有することを特徴とする飼料である。ＦＥＲＭ Ｐ−２０５０１等を用いて基本飼料を発酵させた飼料であってもよく、適量のＦＥＲＭ Ｐ−２０５０１等を基本飼料に単に添加した飼料であってもよい。 The feed of the present invention is a feed comprising lactic acid bacteria FERM P-20501 or FERM P-20500 classified as Leuconostoc mescentroides. It may be a feed obtained by fermenting a basic feed using FERM P-20501 or the like, or a feed obtained by simply adding an appropriate amount of FERM P-20501 or the like to the basic feed.
本発明の飼料は、ＦＥＲＭ Ｐ−２０５０１等を用いて発酵させた飼料であることが好ましい。発酵させることにより、飼料の抗菌性が高められ、安全な長期保存が可能となるためである。また、乳酸菌の分解酵素等により、飼料の消化吸収性を高めることもできるためである。さらに、ＦＥＲＭ Ｐ−２０５０１等が増殖することにより、スターターとしての乳酸菌量を抑えることができるため、経済的にも好ましい。 The feed of the present invention is preferably a feed fermented using FERM P-20501 or the like. By fermenting, the antibacterial property of the feed is enhanced, and safe long-term storage becomes possible. Moreover, it is because the digestibility and absorption of a feed can also be improved by the degradation enzyme of lactic acid bacteria. Furthermore, since the amount of lactic acid bacteria as a starter can be suppressed by growing FERM P-20501 and the like, it is economically preferable.
ＦＥＲＭ Ｐ−２０５０１等を用いて発酵させた飼料の製造方法は、通常乳酸菌を用いて発酵させる方法であれば、特に限定されるものではない。例えば、基本飼料に、１ｘ１０８〜１ｘ１０１０ＣＦＵ／ｇのＦＥＲＭ Ｐ−２０５０１等を接種したものを、フレキシブルなバック等に密封充填し、常温で１〜１４日間静置すること等により発酵させることができる。発酵の際のバック等の外気温は、特に限定されるものではないが、１０〜３７℃が好ましい。 The production method of the feed fermented using FERM P-20501 is not particularly limited as long as it is a method of fermenting using lactic acid bacteria. For example, fermenting basic feed inoculated with 1 × 10 8 to 1 × 10 10 CFU / g of FERM P-20501 etc. in a flexible bag and leaving it at room temperature for 1 to 14 days Can do. Although outside temperature, such as a bag in the case of fermentation, is not specifically limited, 10-37 degreeC is preferable.
また、該基本飼料は、給餌対象の家畜等に応じて適宜選択されるものであり、通常家畜等の飼料に用いられるものであれば、特に限定されるものではない。市販の飼料等でもよいが、植物性の材料を主として用いるものであることが好ましい。ＦＥＲＭ Ｐ−２０５０１等は漬け物から単離された乳酸菌であるため、及び、ロイコノストック属は植物性材料に適合した乳酸菌であるためである。 Further, the basic feed is appropriately selected according to the livestock to be fed, and is not particularly limited as long as it is normally used for livestock. Although commercially available feeds and the like may be used, it is preferable that vegetable materials are mainly used. This is because FERM P-20501 and the like are lactic acid bacteria isolated from pickles, and Leuconostoc is a lactic acid bacterium suitable for plant materials.
特に、ＦＥＲＭ Ｐ−２０５０１等を用いてビール粕を発酵させた飼料が好ましい。ビール粕は、ビール製造過程において、麦芽等の原材料の糖化処理後に発生する不溶性の副産物である。ビール粕には、麦芽由来の酵素等により細かく分解された糖類やタンパク質等が豊富に含有されているために、ＦＥＲＭ Ｐ−２０５０１等の増殖に適しており、かつ、飼料としても、栄養価・消化吸収性の両方に優れており、好ましい。また、ビール粕の再利用の観点からも好ましい。なお、ＦＥＲＭ Ｐ−２０５０１等による発酵を促進するために、ビール粕に、グルコース等の物質をさらに添加しても良い。 In particular, a feed obtained by fermenting beer lees using FERM P-20501 or the like is preferable. Beer koji is an insoluble by-product generated after saccharification of raw materials such as malt during the beer production process. Beer lees are rich in saccharides and proteins that are finely decomposed by malt-derived enzymes and the like, so they are suitable for the growth of FERM P-20501 and the like. It is excellent in both digestibility and absorbability, which is preferable. Moreover, it is preferable also from a viewpoint of reuse of beer lees. In addition, in order to accelerate | stimulate fermentation by FERM P-20501 etc., you may further add substances, such as glucose, to beer lees.
本発明の飼料は、ＦＥＲＭ Ｐ−２０５０１等を用いて発酵させた発酵物を含有する飼料であることが好ましい。予め製造しておいた該発酵物を、基本飼料に含有させることにより、飼料毎に発酵させるよりも、より簡便に、均質なＦＥＲＭ Ｐ−２０５０１等を含有した飼料を製造することができるためである。該発酵物をそのまま基本飼料に含有させてもよく、該発酵物を希釈したものを基本飼料に含有させてもよい。 The feed of the present invention is preferably a feed containing a fermented material fermented using FERM P-20501 or the like. By including the fermented material produced in advance in the basic feed, it is possible to produce a feed containing homogeneous FERM P-20501 and the like more easily than fermenting each feed. is there. The fermented product may be included in the basic feed as it is, or a diluted product of the fermented product may be included in the basic feed.
ＦＥＲＭ Ｐ−２０５０１等を用いて発酵させた発酵物の製造方法は、通常乳酸菌を用いて発酵させる方法であれば、特に限定されるものではない。例えば、炭素源、窒素源、ミネラル、ビタミン等を含有する培地等に、１ｘ１０８〜１ｘ１０１０ＣＦＵ／ｇのＦＥＲＭ Ｐ−２０５０１等を接種したものを、ＦＥＲＭ Ｐ−２０５０１等を用いて発酵させた飼料と同様に発酵させて製造することができる。該培地等は、特に限定されるものではないが、ビール粕を用いることが好ましく、グルコース等の物質をさらに添加したビール粕を用いることがより好ましい。 The manufacturing method of the fermented material fermented using FERM P-20501 is not particularly limited as long as it is a method of fermenting using lactic acid bacteria. For example, a medium containing a carbon source, nitrogen source, mineral, vitamin, etc., inoculated with 1 × 10 8 to 1 × 10 10 CFU / g of FERM P-20501 or the like was fermented using FERM P-20501 or the like. It can be fermented and produced in the same way as feed. The medium and the like are not particularly limited, but beer koji is preferably used, and beer koji further added with a substance such as glucose is more preferably used.
本発明の飼料に含有されるＦＥＲＭ Ｐ−２０５０１等は、生菌体であってもよく、死菌体であってもよい。 FERM P-20501 and the like contained in the feed of the present invention may be live cells or dead cells.
本発明の飼料中のＦＥＲＭ Ｐ−２０５０１等の含有量は、特に限定されるものではない。基本飼料に凍結乾燥機あるいはスプレードライヤー等で乾燥したＦＥＲＭ Ｐ−２０５０１等の死菌体を添加する場合には、理論値で１ｘ１０５〜１ｘ１０８ＣＦＵ／ｇであることが好ましい。ＦＥＲＭ Ｐ−２０５０１等を用いて発酵させた飼料等の生菌体を含有する飼料の場合は、１ｘ１０６〜１ｘ１０８ＣＦＵ／ｇであることが好ましい。 The content of FERM P-20501 and the like in the feed of the present invention is not particularly limited. When dead cells such as FERM P-20501 dried with a freeze dryer or a spray dryer are added to the basic feed, the theoretical value is preferably 1 × 10 5 to 1 × 10 8 CFU / g. In the case of a feed containing live cells such as a feed fermented using FERM P-20501 or the like, it is preferably 1 × 10 6 to 1 × 10 8 CFU / g.
本発明の飼料に含有される、ＦＥＲＭ Ｐ−２０５０１等の形状や、ＦＥＲＭ Ｐ−２０５０１等を用いて発酵させた発酵物の形状は、通常飼料に含有されるものに用いられる形状であれば、特に限定されるものではない。該形状には、例えば、粉末状、粒状、顆粒状、フレーク状、ペレット状がある。但し、乾燥処理後に粉砕して粉末状にする場合には、乾燥処理は６０〜８０℃の温和な条件で行うことが好ましい。また、粒状や顆粒状にする場合に用いられる賦形剤は、通常飼料の製造に用いられるものであれば、特に限定されるものではない。 The shape of FERM P-20501 and the like contained in the feed of the present invention, and the shape of a fermented product fermented using FERM P-20501, etc., are the shapes used for those normally contained in feeds, It is not particularly limited. Examples of the shape include powder, granule, granule, flake, and pellet. However, when the powder is pulverized after the drying treatment, the drying treatment is preferably performed under mild conditions of 60 to 80 ° C. Moreover, the excipient | filler used when making it granular or granular form will not be specifically limited if it is normally used for manufacture of feed.
本発明の飼料には、通常飼料に添加される様々な物質を添加してもよい。該添加される物質として、例えば、ビタミンやミネラル、抗酸化剤等がある。
給餌対象の家畜等に応じて、様々な物質を配合してもよい。該配合される物質として、例えば、粉末カゼイン、糖蜜等がある。
You may add various substances normally added to a feed to the feed of this invention. Examples of the substance to be added include vitamins, minerals and antioxidants.
Various substances may be blended depending on the livestock to be fed. Examples of the substance to be blended include powdered casein and molasses.
本発明の飼料を給餌する方法は、特に限定されるものではなく、通常用いられる飼料と同様に家畜等に摂食させることができる。ＦＥＲＭ Ｐ−２０５０１等の１日あたりの摂取量も、特に限定されるものではなく、家畜等に自由摂取させることができるが、飼料に対し０．０１％（ｗ／ｖ）以上の添加比率で混合するのが好ましい。 The method of feeding the feed of the present invention is not particularly limited, and can be fed to livestock and the like in the same manner as a commonly used feed. The daily intake of FERM P-20501 is not particularly limited, and can be freely consumed by livestock, etc., but at an addition ratio of 0.01% (w / v) or more to the feed It is preferable to mix.
次に試験例を示して本発明をさらに詳細に説明するが、本発明は以下の試験例に限定されるものではない。 Next, although a test example is shown and this invention is demonstrated further in detail, this invention is not limited to the following test examples.
（試験例１）
まず、ＦＥＲＭ Ｐ−２０５０１を用いてビール粕を発酵させた発酵物を含有する飼料（ＦＥＲＭ Ｐ−２０５０１含有飼料）を作製した。予め常法により１晩培養したＦＥＲＭ Ｐ−２０５０１を、およそ１ｘ１０９ＣＦＵ／ｇとなるように、グルコース、炭酸カルシウム等と共にビール粕に添加し、混合した。６５０ｋｇの該混合物を、フレキシブルコンテナバックに充填し、密封後、常温で１〜１４日間静置することにより、発酵させた。発酵後、フレキシブルコンテナバックから取り出した該混合物を、７０℃で乾燥させた後、粉砕して、粉末状のＦＥＲＭ Ｐ−２０５０１含有発酵物を得た。該ＦＥＲＭ Ｐ−２０５０１含有発酵物には、１．２ｘ１０１０ＣＦＵ／ｇのＦＥＲＭ Ｐ−２０５０１が含有されていた。
(Test Example 1)
First, the feed (FERM P-20501 containing feed) containing the fermented material which fermented beer lees using FERM P-20501 was produced. FERM P-20501 previously cultured overnight by a conventional method was added to beer koji together with glucose, calcium carbonate and the like so as to be about 1 × 10 9 CFU / g and mixed. 650 kg of the mixture was filled in a flexible container bag, sealed, and allowed to stand at room temperature for 1 to 14 days for fermentation. After fermentation, the mixture taken out from the flexible container bag was dried at 70 ° C. and then pulverized to obtain a fermented product containing FERM P-20501 in a powder form. The fermented product containing FERM P-20501 contained 1.2 × 10 10 CFU / g of FERM P-20501.
マウスに、ＦＥＲＭ Ｐ−２０５０１含有飼料を摂食させ、免疫機能の変化を測定した。
具体的には、ＡＩＮ−９３Ｇ粉末食（オリエンタル酵母工業株式会社製）を基本飼料とし、これに前記ＦＥＲＭ−２０５０１含有発酵物を０．０１％又は０．１％となるように加えて、ＦＥＲＭ Ｐ−２０５０１含有飼料とし、これを一週間マウスに自由摂食させた。比較対象として、基本飼料のみを摂食させたマウス群と、基本飼料にエンテロコッカス・フェカリス菌ＥＣ−１２株（コンビ社）を２．５ｘ１０１１ｃｅｌｌｓ／ｇ含有した混合飼料「ラクセルフォース」（兼松株式会社製）を０．０１％又は０．１％となるように加えた飼料を摂食させたマウス群を用いた。
Mice were fed a FERM P-20501-containing feed and changes in immune function were measured.
Specifically, AIN-93G powdered food (Oriental Yeast Co., Ltd.) is used as a basic feed, and the fermented product containing FERM-20501 is added to 0.01% or 0.1% to this, and FERM is added. A feed containing P-20501 was used, and the mice were allowed to eat freely for one week. For comparison, a group of mice fed only the basic feed and a mixed feed “Luxell Force” (Kanematsu Co., Ltd.) containing 2.5 × 10 11 cells / g of Enterococcus faecalis EC-12 strain (Combi) in the basic feed. A group of mice fed with a feed supplemented with 0.01% or 0.1% by company) was used.
一週間摂食後、免疫抑制剤として、デキサメタゾン（ＤＥＸ）を各マウスに、５ｍｇ／ｋｇとなるように腹腔内投与した。ＤＥＸは合成副腎皮質ホルモンとして知られており、副腎皮質ホルモンによるストレス性の免疫抑制のモデルとして用いた。ＤＥＸ投与後１８時間経過後、解剖し、各マウスの免疫機能を測定した。具体的には、ＮＫ細胞活性と貪食能を測定した。 After feeding for one week, dexamethasone (DEX) was intraperitoneally administered to each mouse at 5 mg / kg as an immunosuppressant. DEX is known as a synthetic corticosteroid and was used as a model for stress-induced immunosuppression by corticosteroids. After 18 hours from the administration of DEX, the mice were dissected and the immune function of each mouse was measured. Specifically, NK cell activity and phagocytic ability were measured.
表１は、各試験群の試料中の乳酸菌量と供試験動物数を示したものである。試験群４のみ供試験動物数が５匹であるのは、腹腔内投与失敗により、１匹死亡したためである。なお、マウスは、日本ＳＬＣ株式会社より購入した６週齢の雄性のＢＡＬＢ／ｃマウスを、１週間維持した後、実験に用いた。
Table 1 shows the amount of lactic acid bacteria and the number of test animals in the samples of each test group. The reason why the number of test animals in
ＮＫ細胞活性は、各マウスの脾臓細胞の、標的細胞の細胞死への影響を観察することにより測定した。標的細胞として、マウス胸腺細胞腫由来のＹＡＣ−１細胞を用いた。ＹＡＣ−１細胞は、予め、ＰＫＨ２６ Ｒｅｄ Ｆｌｕｏｒｅｓｃｅｎｔ Ｃｅｌｌ Ｌｉｎｋｅｒ Ｋｉｔ (ＳＩＧＭＡ社製)を用いて蛍光標識し、細胞培養液（ＡＩＭ−Ｖ、ＧＩＢＣＯ社製）を用いて２×１０５ｃｅｌｌｓ／ｍＬの細胞浮遊液に調製した。
まず、マウスから摘出した脾臓を、該細胞培養液を用いて洗浄・濾過した後、１×１０７ｃｅｌｌｓ／ｍＬの脾臓細胞浮遊液を調製した。該脾臓細胞浮遊液と、該ＰＫＨ２６標識ＹＡＣ−１細胞浮遊液を、１：５０の割合になるように混合した混合細胞浮遊液を、９６穴平底培養プレート(住友ベークライト株式会社製)に播き、３７℃、５％ＣＯ２条件下で４時間培養した。コントロールとして、ＰＫＨ２６標識ＹＡＣ−１細胞浮遊液のみを９６穴平底培養プレートに播き、同様に培養した。
該混合細胞浮遊液に、蛍光色素ＴＯＰＲＯ−３(Ｍｏｌｅｃｕｌａｒ Ｐｒｏｂｅｓ社製)を添加して死細胞を染色した後、ＢＤ ＦＡＣＳＣａｎｔｏ（ベクトン・ディッキンソン社製）を用いてフローサイトメトリーを行い、ＰＫＨ２６標識ＹＡＣ−１細胞の死細胞率を測定した。ＮＫ細胞活性（％）は、ＹＡＣ−１細胞の全死細胞率（％）からＹＡＣ−１細胞の自然死細胞率（％）を差し引くことにより算出した。なお、ＹＡＣ−１細胞の全死細胞率は、該混合細胞浮遊液の死細胞率であり、ＹＡＣ−１細胞の自然死細胞率は、ＰＫＨ２６標識したＹＡＣ−１細胞浮遊液のみで培養した場合の死細胞率である。
The NK cell activity was measured by observing the influence of the spleen cells of each mouse on the cell death of the target cells. As target cells, mouse thymoma-derived YAC-1 cells were used. YAC-1 cells are fluorescently labeled in advance using PKH26 Red Fluorescent Cell Linker Kit (manufactured by SIGMA), and 2 × 10 5 cells / mL cells using cell culture solution (AIM-V, manufactured by GIBCO). Prepared in suspension.
First, the spleen extracted from the mouse was washed and filtered using the cell culture solution, and then a 1 × 10 7 cells / mL spleen cell suspension was prepared. The mixed cell suspension obtained by mixing the spleen cell suspension and the PKH26-labeled YAC-1 cell suspension at a ratio of 1:50 is seeded on a 96-well flat bottom culture plate (Sumitomo Bakelite Co., Ltd.) The cells were cultured at 37 ° C. under 5% CO 2 for 4 hours. As a control, only the PKH26-labeled YAC-1 cell suspension was seeded on a 96-well flat bottom culture plate and cultured in the same manner.
After adding a fluorescent dye TOPRO-3 (manufactured by Molecular Probes) to the mixed cell suspension to stain dead cells, flow cytometry was performed using BD FACSCanto (manufactured by Becton Dickinson), and PKH26-labeled YAC -1 cell death rate was measured. The NK cell activity (%) was calculated by subtracting the natural dead cell rate (%) of YAC-1 cells from the total dead cell rate (%) of YAC-1 cells. Note that the total dead cell rate of YAC-1 cells is the dead cell rate of the mixed cell suspension, and the natural dead cell rate of YAC-1 cells is when cultured only with a PKH26-labeled YAC-1 cell suspension. Of dead cells.
貪食能は、蛍光標識したラテックスビーズを、各マウスの末梢血中の顆粒球に貪食させることにより測定した。
まず、１．５ｍＬチューブに、５μＬの１×１０９ｂｅａｄｓ／ｍＬに調製した蛍光ラテックスビーズ溶液(Polysciences社製)と、４５μＬの眼窩採血により得た新鮮なマウス末梢血を加え、軽く攪拌した後、３７℃で３０分間インキュベートした。さらに、赤血球を破壊するために、４５０μＬの氷冷した赤血球可溶化溶液（ＮＨ４Ｃｌ：８．２９ｇ／Ｌ、ＫＨＣＯ３：１．０ｇ／Ｌ、ＥＤＴＡ・２Ｎａ：０．０３７ｇ／Ｌ）を加えて軽く攪拌した後、ＢＤ ＦＡＣＳＣａｎｔｏを用いてフローサイトメトリーを行い、末梢血中顆粒球の貪食能を測定した。
貪食能は蛍光ラテックスビーズを貪食した顆粒球の存在比（％）と、顆粒球1細胞あたりが貪食したビーズ数（ｂｅａｄｓ）を乗じたものを指標とした。
Phagocytosis was measured by phagocytosing fluorescently labeled latex beads on granulocytes in the peripheral blood of each mouse.
First, add 5 μL of fluorescent latex bead solution (manufactured by Polysciences) prepared in 1 × 10 9 beads / mL and fresh mouse peripheral blood obtained by orbital blood sampling of 45 μL to a 1.5 mL tube. And incubated at 37 ° C. for 30 minutes. Furthermore, in order to destroy erythrocytes, 450 μL of ice-cold erythrocyte lysate solution (NH 4 Cl: 8.29 g / L, KHCO 3 : 1.0 g / L, EDTA · 2Na: 0.037 g / L) was added. After gently stirring, flow cytometry was performed using BD FACSCanto to measure the phagocytic ability of peripheral blood granulocytes.
The phagocytic ability was determined by multiplying the abundance ratio (%) of granulocytes phagocytosed with fluorescent latex beads by the number of beads phagocytosed per granulocyte (beads).
図１はＮＫ細胞活性の測定結果を、図２は顆粒球貪食能の測定結果を、それぞれ示したものである。図中の１〜５は、表１の試験群を示したものである。 FIG. 1 shows the measurement results of NK cell activity, and FIG. 2 shows the measurement results of granulocyte phagocytosis. 1-5 in a figure show the test group of Table 1. FIG.
図１及び２より明らかであるように、ＦＥＲＭ Ｐ−２０５０１含有飼料を摂食させたマウス（試験群２及び３）は、基本飼料のみを摂食させたマウス（試験群１）や、ラクセルフォース含有飼料を摂食させたマウス（試験群４及び５）に比べ、ＮＫ細胞活性と顆粒球貪食能が向上する傾向が観察された。特に顆粒球貪食能は明らかに向上していた。したがって、本発明のＦＥＲＭ Ｐ−２０５０１を用いてビール粕を発酵させた発酵物を含有する飼料には、免疫機能を向上させる効果があり、安全に家畜の感染症を予防することが期待し得ることが明らかである。また、０．０１％という低濃度のＦＥＲＭ Ｐ−２０５０１含有飼料においても、ＮＫ細胞活性と顆粒球貪食能の向上効果が観察されたことから、経済的にも優れていることが分かった。
As is clear from FIGS. 1 and 2, mice fed the FERM P-20501-containing feed (
（試験例２）
次に、ＦＥＲＭ Ｐ−２０５０１の死菌体の乾燥粉末を前記基本飼料に添加した飼料が、免疫機能向上効果を有するかどうかを観察した。
まず、ＦＥＲＭ Ｐ−２０５０１を飼料添加用培地（４．０（ｗ／ｖ）％グルコース、１．０（ｗ／ｖ）％酵母エキス）に接種し、３０℃で２４時間静置して嫌気培養（前培養）した。得られた前培養液を波長６６０ｎｍでの吸光度（ＯＤ６６０）が１．０となるように調製し、該飼料添加用培地に１／１００（ｖ／ｖ）（菌数：１０７ＣＦＵ／ｍＬ）の割合で接種したものを、ｐＨが６．０を維持するように水酸化ナトリウム溶液を用いてｐＨ調整をしながら、３０℃で２４時間、１００ｒｐｍの攪拌培養（本培養）を行った。得られた本培養液を８，０００ｒｐｍ（約１０，０００×ｇ）で２０分間の遠心分離をして集菌し、これを蒸留水で２回洗浄して菌体を得た。この菌体を、用いた飼料添加用培地量の１／１００量の蒸留水で懸濁し、１０５℃で１分間加熱して死菌体懸濁液を得た。該死菌体懸濁液を、凍結乾燥により乾燥処理し、死菌体菌末を得た。該死菌体菌末には、２．８ｘ１０１２ＣＦＵ／ｇのＦＥＲＭ Ｐ−２０５０１が含有されていた。
(Test Example 2)
Next, it was observed whether or not a feed obtained by adding a dry powder of FERM P-20501 dead cells to the basic feed had an immune function improving effect.
First, FERM P-20501 is inoculated into a medium for feed addition (4.0 (w / v)% glucose, 1.0 (w / v)% yeast extract) and left to stand at 30 ° C. for 24 hours for anaerobic culture. (Pre-culture). The obtained preculture solution was prepared so that the absorbance (OD660) at a wavelength of 660 nm was 1.0, and 1/100 (v / v) (the number of bacteria: 10 7 CFU / mL) in the medium for feed addition The culture was stirred at 100 rpm for 24 hours at 30 ° C. (main culture) while adjusting the pH with a sodium hydroxide solution so that the pH was maintained at 6.0. The obtained main culture was centrifuged at 8,000 rpm (about 10,000 × g) for 20 minutes to collect the cells, which were washed twice with distilled water to obtain bacterial cells. This microbial cell was suspended in 1/100 volume distilled water of the feed addition medium used, and heated at 105 ° C. for 1 minute to obtain a dead cell suspension. The dead cell suspension was dried by lyophilization to obtain a dead cell powder. The dead cell body powder contained 2.8 × 10 12 CFU / g of FERM P-20501.
次に、前記基本飼料に、ＦＥＲＭ Ｐ−２０５０１が理論値でそれぞれ、０ＣＦＵ／ｇ、１．０ｘ１０５ＣＦＵ／ｇ、１．０ｘ１０６ＣＦＵ／ｇ、１．０ｘ１０７ＣＦＵ／ｇ、１．０ｘ１０８ＣＦＵ／ｇとなるように死菌体菌末を添加した飼料を、試験例１と同様に、マウスに一週間自由摂食させた。その後、全て試験例１と同様にして、各マウスの免疫機能を測定した。 Next, in the basic feed, FERM P-20501 is a theoretical value of 0 CFU / g, 1.0 × 10 5 CFU / g, 1.0 × 10 6 CFU / g, 1.0 × 10 7 CFU / g, 1.0 × 10 8 , respectively. In the same manner as in Test Example 1, mice were allowed to freely eat a diet supplemented with dead fungus powder so as to be CFU / g for one week. Thereafter, the immune function of each mouse was measured in the same manner as in Test Example 1.
表２は、各試験群の試料中のＦＥＲＭ Ｐ−２０５０１含有量と供試験動物数を示したものである。試験群１のみ供試験動物数が４匹であるのは、腹腔内投与失敗により、２匹死亡したためである。なお、試験群４の１．０ｘ１０７ＣＦＵ／ｇのＦＥＲＭ Ｐ−２０５０１死菌体菌末含有飼料と、試験例１の０．１％ＦＥＲＭ Ｐ−２０５０１含有飼料が、ＦＥＲＭ Ｐ−２０５０１含有量として同等である。
Table 2 shows the FERM P-20501 content and the number of test animals in the samples of each test group. The reason why the number of test animals in
図３はＮＫ細胞活性の測定結果を、図４は顆粒球貪食能の測定結果を、それぞれ示したものである。図中の１〜５は、表２の試験群を示したものである。 FIG. 3 shows the measurement results of NK cell activity, and FIG. 4 shows the measurement results of granulocyte phagocytosis. 1-5 in a figure show the test group of Table 2. FIG.
図３及び４より明らかであるように、飼料中のＦＥＲＭ Ｐ−２０５０１含有量依存的に、ＮＫ細胞活性と顆粒球貪食能が向上する傾向が観察された。但し、顆粒球貪食能は、１．０ｘ１０７ＣＦＵ／ｇ含有飼料が最も活性が高く、１．０ｘ１０８ＣＦＵ／ｇ含有飼料は１．０ｘ１０６ＣＦＵ／ｇ含有飼料と同程度であったことから、至適な濃度があると推測される。 As is clear from FIGS. 3 and 4, NK cell activity and granulocyte phagocytosis tendencies were improved depending on the FERM P-20501 content in the feed. However, the granulocyte phagocytic ability was highest in the feed containing 1.0 × 10 7 CFU / g, and the feed containing 1.0 × 10 8 CFU / g was comparable to the feed containing 1.0 × 10 6 CFU / g. It is estimated that there is an optimal concentration.
試験例１及び２の結果から、試験例１で観察されたＦＥＲＭ Ｐ−２０５０１を用いてビール粕を発酵させた発酵物を含有する飼料が有する家畜の免疫機能向上効果はＦＥＲＭ Ｐ−２０５０１自身による効果であることが明らかである。 From the results of Test Examples 1 and 2, the effect of improving the immune function of the livestock of the feed containing the fermented fermented beer lees using FERM P-20501 observed in Test Example 1 is due to FERM P-20501 itself. It is clear that this is an effect.
本発明の飼料は、公知のロイコノストック属乳酸菌を含有するものと同様に、整腸作用が期待できることに加え、新規乳酸菌により、免疫機能向上効果を有するものであり、安全に家畜等の健康状態を改善できる飼料として、畜産業の分野で利用が可能である。 The feed of the present invention has the effect of improving the immune function by a novel lactic acid bacterium, in addition to the expectation of intestinal regulation, as well as those containing known leuconostoc lactic acid bacteria. As feed that can improve the condition, it can be used in the field of animal husbandry.
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